Immune-mediated diseases of the colon such as inflammatory bowel disease may be related to a loss of tolerance to commensal bacteria. We have found that in the normal setting, colonic tolerance appears to be maintained via the recognition of commensal bacteria by inducible regulatory T (iTreg) cells. Based on our studies of the colonic TCR repertoire, we have generated two novel transgenic lines expressing bacteria- specific iTreg TCRs. This will allow us to directly assess bacteria-specific iTreg versus effector T cell differentiation under various experimental conditions. In Aim 1, we will manipulate the bacterial population and determine their impact on iTreg cell generation using the TCR transgenic lines, as well as on a polyclonal CD4+ T cell population using TCR repertoire analysis. In Aim 2, we will investigate the antigen-presenting cells and signals that are involved in T cell recognition of bacteria and differentiation into iTreg, rather than effector, cells. Finaly, in Aim 3, we will move beyond normal physiology and study the effect of infections as well as genetic mutations associated with IBD on iTreg cell development, addressing the hypothesis that CD4+ T cell tolerance is broken during immune mediated colitis. We believe that understanding the mechanisms which regulate the development and stability of bacteria-specific colonic iTreg cells will be useful for facilitating the development of Treg cell-mediated therapy fr IBD.